1. Field of the Invention
The present invention relates to methods of generating an ex vivo tissue-like arrangement in a bioreactor system capable of supporting continuous production of, and output of cells and tissue and an ex vivo tissue system tissue made therefrom.
2. Discussion of the Background
There is significant interest using both early and lineage committed cells and organized tissues for a variety of therapeutic and research purposes.
In tissue engineering, the goal is to reconstitute fully or partially functioning human tissue in vitro to enable a variety of clinical, investigational and other applications. Several studies have been carried out that are aimed at reconstituting functioning human tissues in vitro.
The hematopoietic system exemplifies the broad range of cells involved in protection of mammalian hosts against pathogens, toxins, neoplastic cells, and other diseases. The hematopoietic system is believed to evolve from a single stem cell, from which all the lineages of the hematopoietic system derive. Hematopoietic cells have been used in human therapy. Methods and apparatuses for culturing precursor hematopoietic cells to obtain desired mature hematopoietic cells have beers described. See, U.S. Pat. Nos. 5,605,822, 5,399,493; 5,437,994; 5,459,069; 5,635,386, 5,670,147 and 5,670,351.
In addition, bioreactor systems have been described in U.S. Pat. No. 6,875,605 and U.S. Pat. No. 6,493,008, these bioreactor systems, however yield poor cell numbers in long-term culturing and fail to generate tissue-like matrices within the bioreactor because the master in which the media was provided did not allow entrapment of non-adherent cells.
A bioreactor system has also been described and presented at the American Society of Hematology held in San Diego Calif. on Dec. 4,-7, 2004 (Abstract published in Blood, vol. 104(11):Nov. 16, 2004). This bioreactor system was composed of a polycarbonate bioreactor body using a nonwoven polyethylene terephthalate matrix in which cord blood derived stem cells or HS-5 cells were cultivated using recirculating or single pass culturing techniques also using an external gas exchanger. The matrix material used as a scaffold for the cultured cells appeared to have limited biocompatibility. The combination of matrix material, method of cell seeding and the way the reactors were incorporated into a nutrient delivery system combined to make the system sub-optimal. Toxicity issues were still apparent in these designs.
These prior devices are unable to support tissue-like culture density due to a poor gas exchange mediated by the gassing membrane, which prevents migration of the cells into the depth of the support matrix, which, in turn, does not permit the establishment of a gradient of media and growth factor availability to the cells mimicking intact human tissues. Still further, as the gas exchange is so poor in these prior devices, increased gas delivery is to be provided by increasing the flow rate of the media with gas being provided though as external source.
Moreover, these prior devices do not generate ex vivo tissue because, the fluid pathway is too thick and requires seeding through a depth filter method, resulting in the removal of non-adherent cells, which significantly impairs the long term ability of the culture to generate a tissue and maintain function. Overall, the general goal of the above methods and other prior methods and devices was to optimize culturing conditions for homogenous cultures of specific cell types (whether mixed with additional support cells or contaminants.)
There remains a need for apparatus that maintains a culture of either homologous or heterogonous cells in an organized arrangement in three-dimensions and allows the development of differing microenvironments conducive to tissue expansion and maintenance for extended periods of time with the ability to change the microenvironment parameters within the culture and which can permit the ex vivo generation of tissue-like matrices of cells.